1. Field of the Invention
The present invention concerns a method for the production of a biochemical selected among acetol and 1,2-propanediol, 1,3-propanediol, ethylene glycol and 1,4-butanediol comprising culturing a microorganism modified for an improved production of the biochemical selected among acetol and 1,2-propanediol, 1,3-propanediol, ethylene glycol and 1,4-butanediol in an appropriate culture medium and recovery of the desired biochemical which may be further purified wherein the microorganism expresses a YqhD enzyme which catalytic efficiency toward NADPH is increased.
The present invention also relates to a mutant YqhD enzyme comprising at least one amino acid residue in the protein sequence of the parent enzyme replaced by a different amino acid residue at the same position wherein                the mutant enzyme has retained more than 50% of the YqhD activity of the parent enzyme and        the catalytic efficiency toward NADPH of the mutant YqhD is increased as compared with the catalytic efficiency toward NADPH of the parent enzyme.        
1. Description of Related Art
YqhD was first identified as an 1,3-propanediol oxidoreductase catalysing the NADPH-dependent reduction of 3-hydroxypropionaldehyde (3-HPA) into 1,3-propanediol (PDO). This activity, endogenous to E. coli, was found to be more efficient that the recombinant NADH-dependent activity (encoded by dhaT gene from Klebsiella pneumoniae) used to built a PDO biosynthetic pathway in E. coli (Nakamura and Whited, 2003). The Du Pont de Nemours—Genencor project for the production of PDO in E. coli resulted in an industrial process with high titers and high yields (WO2001/012833 and WO2004/033646).
Expression of the yqhD gene was later used for purpose of production of PDO in different organisms, E. coli or Saccharomyces cerevisiae (Wang et al, 2007, Rao et al, 2008, Wang et al, 2009).
We showed that YqhD can act as a methylglyoxal reductase in the biosynthesis of 1,2-propanediol in E. coli (WO2008/116853).
The crystal structure of YqhD was determined with bound NADP co-factor. YqhD was shown to be a tetramer containing 1 atom of Zn by monomer. Alcohol dehydrogenase activity was recorded on several alcohols but with a weak affinity. However, the discovery of the presence of a modified (hydroxylated) co-factor in the enzyme cast doubt about the activity data provided (Sulzenbacher et al, 2004).
A more recent study characterized YqhD as an NADPH-dependent aldehyde reductase working on different substrates: acetaldehyde, propanaldehyde, butanaldehyde, acrolein and malondialdehyde (Perez et al, 2008). In this study, no activity was detected on short or medium chain alcohols. These authors proposed that YqhD was of relevance for the detoxification of toxic aldehydes inside the cell.
Miller et al (2009) showed that purified YqhD exhibited a furfural reductase activity. In this study, YqhD was found to have a very strong affinity for its NADPH co-factor with Km value for NADPH of 8 μM. With such an affinity, an active YqhD could scavenge NADPH normally used for the biosynthetic reactions of the cell and would thus inhibit growth.
Mutants of YqhD were produced by error-prone PCR in the aim of improving the enzyme activity towards 3-HPA for the production of 1,3-propanediol (Li et al, 2008). Two mutants, a double mutant (D99Q N147H) and a single mutant ((Q202A) were found to have an improved affinity (lower Km) for 3-HPA and an improved catalytic efficiency (Kcat/Km) for 3-HPA.
PDO is a monomer used in the production of polyester fibers, especially poly(trimethylene terephthalate) (PTT) and with potential applications in the manufacture of polyurethanes and cyclic compounds.
PDO can be produced by different chemical routes from i) acrolein, water and hydrogen, ii) ethylene oxide, carbon monoxide and water in the presence of phosphine and iii) glycerol and hydrogen in the presence of carbon monoxide. All of these methods have in common to be expensive and to generate waste streams containing toxic substances potentially harmful to the environment.
PDO is a typical product of glycerol fermentation and has not been found in anaerobic conversions of other organic substrates. Only very few organisms, all of them bacteria, are able to form it. They include enterobacteria of the genera Klebsiella (K. pneumoniae), Enterobacter (E. agglomerans) and Citrobacter (C. freundii), lactobacilli (L. brevis and L. buchneri) and clostridia of the C. butyricum and the C. pasteurianum group.
New bioprocesses for the production of PDO in recombinant E. coli have been disclosed in WO2001/012833 and WO2004/033646. These processes rely on the utilization of the yqhD gene encoding the YqhD enzyme for the last step of synthesis of PDO. Production of PDO from glycerol in a recombinant Klebsiella expressing the yqhD gene was described in CN1011260379. The same system applied to the production of PDO from glucose in a recombinant Saccharomyces cerevisiae was disclosed in CN101130782.
An alternative solution to produce PDO in recombinant organisms using a different biosynthetic pathway without glycerol as an intermediate has been proposed in patent application EP08173129.1. Production of other diols, such as ethylene glycol and 1,4-butanediol, according to the same scheme was also incorporated in the same patent application. All these processes are using the enzyme YqhD for the last step of synthesis.
1,2-propanediol or propylene glycol, a C3 dialcohol, is a widely-used chemical. It is a component of unsaturated polyester resins, liquid detergents, coolants, anti-freeze and de-icing fluids for aircraft. Propylene glycol has been increasingly used since 1993-1994 as a replacement for ethylene derivatives, which are recognised as being more toxic than propylene derivatives.
1,2-propanediol is currently produced by chemical means using a propylene oxide hydration process that consumes large amounts of water. Propylene oxide can be produced by either of two processes, one using epichlorhydrin, and the other hydroperoxide. Both routes use highly toxic substances. In addition, the hydroperoxide route generates by-products such as tert-butanol and 1-phenyl ethanol. For the production of propylene to be profitable, a use must be found for these by-products. The chemical route generally produces racemic 1,2-propanediol, whereas each of the two stereoisomers (R)1,2-propanediol and (S)1,2-propanediol are of interest for certain applications (e.g. chiral starting materials for specialty chemicals and pharmaceutical products).
Acetol or hydroxyacetone (1-hydroxy- 2-propanone) is a C3 keto alcohol. This product is used in vat dyeing process in the textile industry as a reducing agent. It can advantageously replace traditional sulphur containing reducing agents in order to reduce the sulphur content in wastewater, harmful for the environment. Acetol is also a starting material for the chemical industry, used for example to make polyols or heterocyclic molecules. It possesses also interesting chelating and solvent properties.
Acetol is currently produced mainly by catalytic oxidation or dehydration of 1,2-propanediol. New processes starting from renewable feedstocks like glycerol are now proposed (see DE4128692 and WO 2005/095536). Currently, the production cost of acetol by chemical processes reduces its industrial applications and markets.
The disadvantages of the chemical processes for the production of 1,2-propanediol and acetol make biological synthesis an attractive alternative. 1,2-propanediol can be derived from central metabolism in three steps whereas acetol can be obtained in two steps. Methylglyoxal synthase converting dihydroxyacetone phosphate into methylglyoxal (MG) is the mandatory first step for the production of these two compounds. MG can then be converted to either acetol or D- or L-lactaldehyde by methylglyoxal reductases (Cameron et al, 1998, Bennett and San, 2001, Ko et al, 2005). As mentioned earlier, YqhD was shown to be very efficient in the production of acetol from methylglyoxal (WO2008/116853). Acetol or lactaldehyde can be converted to 1,2-propanediol by several enzymatic activities, especially glycerol dehydrogenase (encoded by gldA gene) or 1,2-propanediol oxidoreductase (encoded by fucO gene) in E. coli (Altaras and Cameron, 2000).
Processes for the production of 1,2-propanediol or acetol using different microorganisms, Clostridium sphenoides (DE3336051), Klebsiella pneumoniae (WO 2004/087936), recombinant yeast (WO 99/28481) or recombinant E. coli (WO 98/37204) have been disclosed. We recently proposed alternative approaches for the production of 1,2-propanediol or acetol (WO 2005/073364, WO 2008/116852, WO 2008/116848, WO 2008/116849, WO 2008/116851)), some of them relying on the use of the methylglyoxal reductase YqhD.
During their investigations on 1,2-propanediol production, the inventors identified new mutant YqhD enzymes with increased catalytic efficiency (increased Kcat/Km) toward NADPH, while keeping most of their specific activity for the conversion of methylglyoxal into acetol, as demonstrated in Example 2 by the characterization of purified enzymes. Use of these mutants is a key element in the design of more efficient processes for the production of all the products using a metabolic pathway based on YqhD activity.